Evaluation of Multiplex Immunoassay Results

Cost- and Time-Effective Method Must Be Selected with Care to Ensure Accuracy

Fluorokine® Multi-Analyte Profiling (Fluorokine MAP) kits from R&D Systems are designed for the simultaneous detection and quantification of up to 100 analytes and provide a highly efficient means for profiling process-related changes in the levels of biomolecules. Measurement is achieved through a bead-based antibody-antigen sandwich method similar to that used in single analyte plate based ELISAs.

Using a Luminex®-based analyzer, beads are classified by their unique fluorescent signatures and quantified by the magnitude of the PE-derived signals that are directly proportional to the levels of bound analyte.

Many researchers have taken advantage of the efficiency and flexibility afforded by commercially available Fluorokine MAP kits. These kits offer bead sets, detection antibodies, and associated reagents for measuring the levels of cytokines, growth factors, chemokines, matrix metalloproteinases, obesity/diabetes-related factors, angiogenesis factors, and more.

Since their inception, there has been an expectation that multiplex technologies produce quantitative results similar to those of a single analyte ELISA. However, the possibility of cross-reactivity and interference multiplies with the complexity of each panel. Fluorokine MAP kits must be developed and optimized to achieve accurate and reproducible results for all analytes in the panel using a single set of multipurpose diluents.

These multipurpose diluents may not optimize any single analyte to the same degree that is possible with a unique diluent selected for a single analyte ELISA. However, the accuracy of the assay must not be compromised.

This article highlights why the results for each analyte, and the panel as a whole, should be evaluated as rigorously as a single analyte ELISA. This includes assessing specificity, recovery, and linearity of dilution, in addition to sensitivity, precision, and the ability to recognize the natural proteins. Only in this way can results that are comparable to those of a single analyte ELISA be achieved.

Michael Anderson (michael.anderson@rndsystems.com) is manager of new technologies, James David is team leader, new technologies, Samuel Pearlman is director of human assay development, and Jane Schmidt, Ph.D., is director of new technologies at R&D Systems.

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